The objective of this proposal is to investigate the mechanism of ganciclovir cytotoxicity and the "bystander effect" in two human colon carcinoma cell lines (HT29, SW620) expressing herpes simplex virus thymidine kinase. Preliminary experiments revealed differential sensitivity to ganciclovir between cell lines, as well as divergent capacities for intercellular communication. The initial aim will define which elements are responsible for the cytotoxic activity of ganciclovir and determine why one cell line is more or less sensitive to this therapy. A comparison between these two cell lines will be used to determine a relationship between ganciclovir metabolism, its consequences on DNA synthesis and integrity and its effect on bystander killing. Subsequent studies will focus on enhancing bystander killing by manipulating HSV-tk expression, endogenous dNTP pools and gap junctional intercellular communication.